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Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Metagenome studies have provided us with insights into the complex interactions of microorganisms with their environments and hosts. Few studies have focused on microalgae-associated metagenomes, and no study has addressed aquatic microalgae and their bacterial communities in open pond raceways (OPRs). This study explored the possibility of using microalgal biomasses from OPRs for biodiesel and biofertilizer production. The fatty acid profiles of the biomasses and the physical and chemical properties of derived fuels were evaluated. In addition, the phenotype-based environmental adaptation ability of soybean plants was assessed. The growth rate, biomass, and lipid productivity of microalgae were also examined during mass cultivation from April to November 2017. Metagenomics analysis using MiSeq identified ∼127 eukaryotic phylotypes following mass cultivation with (OPR 1) or without (OPR 3) a semitransparent film. Of these, ∼80 phylotypes were found in both OPRs, while 23 and 24 phylotypes were identified in OPRs 1 and 3, respectively. The phylotypes belonged to various genera, such as Desmodesmus, Pseudopediastrum, Tetradesmus, and Chlorella, of which, the dominant microalgal species was Desmodesmus sp. On average, OPRs 1 and 3 produced ∼8.6 and 9.9 g m−2 d−1 (0.307 and 0.309 DW L−1) of total biomass, respectively, of which 14.0 and 13.3 wt% respectively, was lipid content. Fatty acid profiling revealed that total saturated fatty acids (mainly C16:0) of biodiesel obtained from the microalgal biomasses in OPRs 1 and 3 were 34.93% and 32.85%, respectively; total monounsaturated fatty acids (C16:1 and C18:1) were 32.40% and 31.64%, respectively; and polyunsaturated fatty acids (including C18:3) were 32.68% and 35.50%, respectively. Fuel properties determined by empirical equations were within the limits of biodiesel standards ASTM D6751 and EN 14214. Culture solutions with or without microalgal biomasses enhanced the environmental adaptation ability of soybean plants, increasing their seed production. Therefore, microalgal biomass produced through mass cultivation is excellent feedstock for producing high-quality biodiesel and biofertilizer.

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Background Similar to natural rivers, manmade inlets connect inland runoff to the ocean. Port Everglades Inlet (PEI) is a busy cargo and cruise ship port in South Florida, which can act as a source of pollution to surrounding beaches and offshore coral reefs. Understanding the composition and fluctuations of bacterioplankton communities (“microbiomes”) in major port inlets is important due to potential impacts on surrounding environments. We hypothesize seasonal microbial fluctuations, which were profiled by high throughput 16S rRNA amplicon sequencing and analysis. Methods & Results Surface water samples were collected every week for one year. A total of four samples per month, two from each sampling location, were used for statistical analysis creating a high sampling frequency and finer sampling scale than previous inlet microbiome studies. We observed significant differences in community alpha diversity between months and seasons. Analysis of composition of microbiomes (ANCOM) tests were run in QIIME 2 at genus level taxonomic classification to determine which genera were differentially abundant between seasons and months. Beta diversity results yielded significant differences in PEI community composition in regard to month, season, water temperature, and salinity. Analysis of potentially pathogenic genera showed presence of Staphylococcus and Streptococcus. However, statistical analysis indicated that these organisms were not present in significantly high abundances throughout the year or between seasons. Discussion Significant differences in alpha diversity were observed when comparing microbial communities with respect to time. This observation stems from the high community evenness and low community richness in August. This indicates that only a few organisms dominated the community during this month. August had lower than average rainfall levels for a wet season, which may have contributed to less runoff, and fewer bacterial groups introduced into the port surface waters. Bacterioplankton beta diversity differed significantly by month, season, water temperature, and salinity. The 2013–2014 dry season (October–April), was warmer and wetter than historical averages. This may have driven significant differences in beta diversity. Increased nitrogen and phosphorous concentrations were observed in these dry season months, possibly creating favorable bacterial growth conditions. Potentially pathogenic genera were present in the PEI. However their relatively low, non-significant abundance levels highlight their relatively low risk for public health concerns. This study represents the first to sample a large port at this sampling scale and sequencing depth. These data can help establish the inlet microbial community baseline and supplement the vital monitoring of local marine and recreational environments, all the more poignant in context of local reef disease outbreaks and worldwide coral reef collapse in wake of a harsh 2014–16 El Niño event. [ABSTRACT FROM AUTHOR]

Background Similar to natural rivers, manmade inlets connect inland runoff to the ocean. Port Everglades Inlet (PEI) is a busy cargo and cruise ship port in South Florida, which can act as a source of pollution to surrounding beaches and offshore coral reefs. Understanding the composition and fluctuations of bacterioplankton communities (“microbiomes”) in major port inlets is important due to potential impacts on surrounding environments. We hypothesize seasonal microbial fluctuations, which were profiled by high throughput 16S rRNA amplicon sequencing and analysis. Methods & Results Surface water samples were collected every week for one year. A total of four samples per month, two from each sampling location, were used for statistical analysis creating a high sampling frequency and finer sampling scale than previous inlet microbiome studies. We observed significant differences in community alpha diversity between months and seasons. Analysis of composition of microbiomes (ANCOM) tests were run in QIIME 2 at genus level taxonomic classification to determine which genera were differentially abundant between seasons and months. Beta diversity results yielded significant differences in PEI community composition in regard to month, season, water temperature, and salinity. Analysis of potentially pathogenic genera showed presence of Staphylococcus and Streptococcus. However, statistical analysis indicated that these organisms were not present in significantly high abundances throughout the year or between seasons. Discussion Significant differences in alpha diversity were observed when comparing microbial communities with respect to time. This observation stems from the high community evenness and low community richness in August. This indicates that only a few organisms dominated the community during this month. August had lower than average rainfall levels for a wet season, which may have contributed to less runoff, and fewer bacterial groups introduced into the port surface waters. Bacterioplankton beta diversity differed significantly by month, season, water temperature, and salinity. The 2013–2014 dry season (October–April), was warmer and wetter than historical averages. This may have driven significant differences in beta diversity. Increased nitrogen and phosphorous concentrations were observed in these dry season months, possibly creating favorable bacterial growth conditions. Potentially pathogenic genera were present in the PEI. However their relatively low, non-significant abundance levels highlight their relatively low risk for public health concerns. This study represents the first to sample a large port at this sampling scale and sequencing depth. These data can help establish the inlet microbial community baseline and supplement the vital monitoring of local marine and recreational environments, all the more poignant in context of local reef disease outbreaks and worldwide coral reef collapse in wake of a harsh 2014–16 El Niño event.

Microsatellite markers remain an important tool for ecological and evolutionary research, but are unavailable for many non-model organisms. One such organism with rare ecological and evolutionary features is the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1758). Chelonibia testudinaria appears to be a host generalist, and has an unusual sexual system, androdioecy. Genetic studies on host specificity and mating behavior are impeded by the lack of fine-scale, highly variable markers, such as microsatellite markers. In the present study, we discovered thousands of new microsatellite loci from next-generation sequencing data, and characterized 12 loci thoroughly. We conclude that 11 of these loci will be useful markers in future ecological and evolutionary studies on C. testudinaria.

In this study, we sequenced the genome of Pandoraea pnomenusa RB38 using Pacific Biosciences RSII (PacBio) Single Molecule Real Time (SMRT) sequencing technology. A pair of cognate luxI/R homologs was identified where the luxI homolog, ppnI, was found adjacent to a luxR homolog, ppnR1. An additional orphan luxR homolog, ppnR2, was also discovered. Multiple sequence alignment and phylogenetic analysis revealed that ppnI is an N-acyl homoserine lactone (AHL) synthase gene that is distinct from those of the nearest phylogenetic neighbor viz. Burkholderia spp. High resolution tandem mass spectrometry (LC-MS/MS) analysis showed that Escherichia coli BL21 harboring ppnI produced a similar AHL profile (N-octanoylhomoserine lactone, C8-HSL) as P. pnomenusa RB38, the wild-type donor strain, confirming that PpnI directed the synthesis of AHL in P. pnomenusa RB38. To our knowledge, this is the first documentation of the luxI/R homologs of the genus Pandoraea.

Metagenome studies have provided us with insights into the complex interactions of microorganisms with their environments and hosts. Few studies have focused on microalgae-associated metagenomes, and no study has addressed aquatic microalgae and their bacterial communities in open pond raceways (OPRs). This study explored the possibility of using microalgal biomasses from OPRs for biodiesel and biofertilizer production. The fatty acid profiles of the biomasses and the physical and chemical properties of derived fuels were evaluated. In addition, the phenotype-based environmental adaptation ability of soybean plants was assessed. The growth rate, biomass, and lipid productivity of microalgae were also examined during mass cultivation from April to November 2017. Metagenomics analysis using MiSeq identified ∼127 eukaryotic phylotypes following mass cultivation with (OPR 1) or without (OPR 3) a semitransparent film. Of these, ∼80 phylotypes were found in both OPRs, while 23 and 24 phylotypes were identified in OPRs 1 and 3, respectively. The phylotypes belonged to various genera, such as Desmodesmus , Pseudopediastrum , Tetradesmus , and Chlorella , of which, the dominant microalgal species was Desmodesmus sp. On average, OPRs 1 and 3 produced ∼8.6 and 9.9 g m -2 d -1 (0.307 and 0.309 DW L -1 ) of total biomass, respectively, of which 14.0 and 13.3 wt% respectively, was lipid content. Fatty acid profiling revealed that total saturated fatty acids (mainly C16:0) of biodiesel obtained from the microalgal biomasses in OPRs 1 and 3 were 34.93% and 32.85%, respectively; total monounsaturated fatty acids (C16:1 and C18:1) were 32.40% and 31.64%, respectively; and polyunsaturated fatty acids (including C18:3) were 32.68% and 35.50%, respectively. Fuel properties determined by empirical equations were within the limits of biodiesel standards ASTM D6751 and EN 14214. Culture solutions with or without microalgal biomasses enhanced the environmental adaptation ability of soybean plants, increasing their seed production. Therefore, microalgal biomass produced through mass cultivation is excellent feedstock for producing high-quality biodiesel and biofertilizer., Competing Interests: The authors declare there are no competing interests., (©2020 Jo et al.)

Background: Similar to natural rivers, manmade inlets connect inland runoff to the ocean. Port Everglades Inlet (PEI) is a busy cargo and cruise ship port in South Florida, which can act as a source of pollution to surrounding beaches and offshore coral reefs. Understanding the composition and fluctuations of bacterioplankton communities ("microbiomes") in major port inlets is important due to potential impacts on surrounding environments. We hypothesize seasonal microbial fluctuations, which were profiled by high throughput 16S rRNA amplicon sequencing and analysis., Methods & Results: Surface water samples were collected every week for one year. A total of four samples per month, two from each sampling location, were used for statistical analysis creating a high sampling frequency and finer sampling scale than previous inlet microbiome studies. We observed significant differences in community alpha diversity between months and seasons. Analysis of composition of microbiomes (ANCOM) tests were run in QIIME 2 at genus level taxonomic classification to determine which genera were differentially abundant between seasons and months. Beta diversity results yielded significant differences in PEI community composition in regard to month, season, water temperature, and salinity. Analysis of potentially pathogenic genera showed presence of Staphylococcus and Streptococcus . However, statistical analysis indicated that these organisms were not present in significantly high abundances throughout the year or between seasons., Discussion: Significant differences in alpha diversity were observed when comparing microbial communities with respect to time. This observation stems from the high community evenness and low community richness in August. This indicates that only a few organisms dominated the community during this month. August had lower than average rainfall levels for a wet season, which may have contributed to less runoff, and fewer bacterial groups introduced into the port surface waters. Bacterioplankton beta diversity differed significantly by month, season, water temperature, and salinity. The 2013-2014 dry season (October-April), was warmer and wetter than historical averages. This may have driven significant differences in beta diversity. Increased nitrogen and phosphorous concentrations were observed in these dry season months, possibly creating favorable bacterial growth conditions. Potentially pathogenic genera were present in the PEI. However their relatively low, non-significant abundance levels highlight their relatively low risk for public health concerns. This study represents the first to sample a large port at this sampling scale and sequencing depth. These data can help establish the inlet microbial community baseline and supplement the vital monitoring of local marine and recreational environments, all the more poignant in context of local reef disease outbreaks and worldwide coral reef collapse in wake of a harsh 2014-16 El Niño event., Competing Interests: The authors declare there are no competing interests.

Microsatellite markers remain an important tool for ecological and evolutionary research, but are unavailable for many non-model organisms. One such organism with rare ecological and evolutionary features is the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1758). Chelonibia testudinaria appears to be a host generalist, and has an unusual sexual system, androdioecy. Genetic studies on host specificity and mating behavior are impeded by the lack of fine-scale, highly variable markers, such as microsatellite markers. In the present study, we discovered thousands of new microsatellite loci from next-generation sequencing data, and characterized 12 loci thoroughly. We conclude that 11 of these loci will be useful markers in future ecological and evolutionary studies on C. testudinaria.

In this study, we sequenced the genome of Pandoraea pnomenusa RB38 using Pacific Biosciences RSII (PacBio) Single Molecule Real Time (SMRT) sequencing technology. A pair of cognate luxI/R homologs was identified where the luxI homolog, ppnI, was found adjacent to a luxR homolog, ppnR1. An additional orphan luxR homolog, ppnR2, was also discovered. Multiple sequence alignment and phylogenetic analysis revealed that ppnI is an N-acyl homoserine lactone (AHL) synthase gene that is distinct from those of the nearest phylogenetic neighbor viz. Burkholderia spp. High resolution tandem mass spectrometry (LC-MS/MS) analysis showed that Escherichia coli BL21 harboring ppnI produced a similar AHL profile (N-octanoylhomoserine lactone, C8-HSL) as P. pnomenusa RB38, the wild-type donor strain, confirming that PpnI directed the synthesis of AHL in P. pnomenusa RB38. To our knowledge, this is the first documentation of the luxI/R homologs of the genus Pandoraea.